Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ

in size, charge or conformation.

ELECTROPHORESIS:

GEL ELECTROPHORESIS; is used to sort protein strands by length.

Gel is a filter like a sponge to separate DNA

PRINCIPLE:

When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode

PROCEDURE :1. To prepare 100 ml of a 0.7% agarose solution, measure 0.7 g agarose into a glass beaker or flask and add 100 ml 1X buffer. Microwave or stir on a hot plate until agarose is dissolved and solution is clear. 2. Allow solution to cool to about 55 degrees C before pouring. (Ethidium bromide can be added at this point to a concentration of 0.5 g/ml) 3. Prepare gel tray by sealing ends with tape or other custom-made dam. Place comb in gel tray about 1 inch from one end of the tray and position the comb vertically such that the teeth are about 1-2 mm above the surface of the tray 4. Pour 50 degree C gel solution into tray to a depth of about 5 mm. Allow gel to solidify about 20 minutes at room temperature. 5. To run, gently remove the comb, place tray in electrophoresis chamber, and cover (just until wells are submerged) with electrophoresis buffer (the same buffer used to prepare the agarose). Excess agarose can be stored at room temperature and remelted in a microwave. To prepare samples for electrophoresis, add 1 l of 6x gel

loading dye for every 5 l of PROTEIN solution. Mix well. Load 5-12 l of PROTEIN per well (for minigel). Electrophorese at 50-150 volts until dye markers have migrated an appropriate distance, depending on the size of PROTEIN to be visualized. 6. If the gel was not stained with ethidium during the run, stain the gel in 0.5 g/ml ethidium bromide until the PROTEIN has taken up the dye and is visible under short wave UV light, if the PROTEIN will not be used further, or with a hand-held long-wave UV light if the PROTEIN is to be cut out and purified.

TWO DIMENSIONAL GEL ELECTROPHORESIS:

Two-dimensional polyacrylamide gel electrophoresis (PAGE) is used for separation of complex protein mixtures by the independent parameters of isoelectric point and molecular weight

PRINCIPLE:Isoelectric focusing (IEF) separates proteins in a pH gradient. Each protein is 'focused' because it moves under the influence of the electric field until it reaches its isoelectric point, the pH at which it has no net charge. After IEF in the presence of urea and a nonionic detergent, the IEF gel is equilibrated in sodium dodecyl sulfate (SDS) to prepare the proteins for SDS-PAGE.

MATERIAL AND REAGENTS :

ISOELECTRIC FOCUSING/ THE FIRST DIMENSION

1) Prepare 2.5mL of fresh rehydration buffer for each type of Dry Strip. 2) Place samples on ice to thaw and collect 1.5mL microcentrifuge tubes for sample dilution. 3) Remove the necessary number of Dry Strips from -20C freezer. 4) Determine the amount of rehydration buffer necessary for strip length. 5) Add 200-300ug of cell lysate to clean micro centrifuge tubes and add the difference between the total volume and the volume of sample using fresh rehydration buffer.

6) Remove plastic cover from strip holder and set aside 7) Add rehydration buffer containing sample to the strip holder at the anode (pointed end of the holder). 8) Remove protective cover from DryStrip before placing gel side down into strip holder starting at the anode (pointed end) and laying strip down to the cathode (blunt end). 9) Move strip back and forth in order to spread out rehydration buffer. 10) Make sure that all bubbles are removed from underneath the DryStrip before adding Dry Strip Cover Fluid (Mineral Oil). 11) Add cover fluid from one end until it reaches the middle of the strip holder then add from the opposite side so that fluid meets in the middle. 12) Complete the same process for all strips then place the strip holders on IPGphor system. 13) Set up protocol for the length strip that is being us 14) For 13cm strip: Rehydration at 20C for 12 hours 50uA per strip 500V for 1 hour 1000V for 1 hour 8000V for 3-5 hours 15) Move onto equilibration step or rinse strip with MilliQ water and place into screw cap Tubes for storing in -70C freezer

RUNNING THE SECOND DIMENSION:

1) Prepare gel by removing water saturated butanol from top of gel and washing with 1X running buffer. 2) Cover top of gel with 1X running buffer and lay strip across the top of the gel making sure that the gel is lying flush with the gel and remove any bubbles between the strip and the top of the gel. 3) Remove the running buffer and add warm 1% agarose made with 1X running buffer. 4) Allow the agarose to cool and solidify, which should only take a few minutes, before moving to the electrophoresis apparatus. 5) Add 1X running buffer to the upper and lower buffer chambers and place gel inside apparatus. 6) Run gel for 15 minutes at 10mA per gel then run gel at 25mA per gel for 4-6 hours (until BPB band reaches bottom of gel). 7) Remove gel from plates and stain

GEL VISUALISATION:

Copper Staining 1. Prepare the Copper stain by dissolving 4 g of CuCl2 in 100 mL of water. 2. Wash the gels with nanopure water. 3. Pour the Cu stain on the gel. Make sure that the gel is completely submerged. 4. Rock for 5 minutes. 5. Pour the stain into the waste and wash the gel with water to remove excess copper. 6. Store the gel in water in the refrigerator. ZInC Staining mix thoroughly. Dilute Zinc Sulfate (Solution B) 1:10 with deionized water, mix thoroughly. Gels will require 100-150 mL so that they are compeletely immersed. 2. Place the gel in a staining container and add Imidazole, solution A. Rock at room temperature for 10 min. A, and add the dilute Zinc sulfate, solution B. Make sure the gel is completely covered to ensure even staining. Rock at room temperature for 30 sec approx while the gel develops.4. Decant solution B, and add 100 mL of water. Rinse for 3-5 min rocking at room temperature 3. Decant Solution 1. Dilute Imidazole (Solution A) 1:10 with water,

5. Decant water and replace with fresh water. The gel can be stored like this for days.